Abstract

We describe the corkscrew point spread function (PSF), which can localize objects in three dimensions throughout a 3.2μm depth of field with nanometer precision. The corkscrew PSF rotates as a function of the axial (z) position of an emitter. Fisher information calculations show that the corkscrew PSF can achieve nanometer localization precision with limited numbers of photons. We demonstrate three-dimensional super-resolution microscopy with the corkscrew PSF by imaging beads on the surface of a triangular polydimethylsiloxane (PDMS) grating. With 99,000 photons detected, the corkscrew PSF achieves a localization precision of 2.7nm in x, 2.1nm in y, and 5.7nm in z.

Figures (4)

(a) Images of the corkscrew PSF collected from a fluorescent bead at various z positions. Scale bars are 1μm. (b) 3D isosurface rendering of the corkscrew PSF, where the z positions of the blue planes correspond to the cross sections in (a). (c) Angle (extracted from pairs of images) versus z calibration curve measured at discrete z positions. The corkscrew PSF smoothly rotates 330 degrees over a 3.2μm depth of field.

(a) Composite image of fluorescent beads on a triangular PDMS grating using the original (red) and rotated (cyan) versions of the corkscrew PSF. The height variations of the grating are clearly visible from the various orientations of the PSF. Inset shows a corresponding white-light image of the grating. The flat tops and large height of the grating, combined with tilted illumination, produce shadows in the image. Axis arrows are 3μm. (b) Locations of beads (red circles, diameter=0.2μm) over multiple periods of the grating, projected onto the yz plane. Gray line is a model of the grating (3.0μm periodicity, 1.8μm height). Note the good agreement between bead locations and the grating model. Axis arrows are 1μm. (c) Bead locations from multiple periods of the grating combined together, showing the translational symmetry of the measurement. Axis arrows are 1μm.